Exercise 8.3 - The Hill Reaction

LEVEL I

Materials

• Chloroplast suspension (Exercise 8.2)
  
• 0.035 M NaCl
  
• Boiling water bath
  
• Ice bath
  
• 5 x 10 M 3-(3,4-Dichlorophenyl)-1,1,-Dimethylurea (DCMU) 3
  
• 0.1 M KHPO/KHPO Buffer, pH 6.5
  
• 1 x 10 M Dichlorophenolindophenol (DCPIP) 4

Procedure

1. Dilute the chloroplast suspension with 0.035 M NaCl to yield a final chlorophyll concentration of 0.01 mg/ml (Refer to Exercise 8.2).

2. Heat a 5.0 ml portion of the diluted suspension into a boiling water bath. Remove the inactivated chloroplasts from the boiling water after five minutes and place in an ice bath to cool.

   Keep the remainder of the active chloroplast suspension at 4° C (ice bath).

3. Prepare five test tubes as follows:

   	1	2	3	4	5
   Buffer	3.0 ml	3.0 ml	3.0 ml	3.0 ml	3.0 ml
   Heated chloroplasts	1.0 ml	1.0 ml	None	None	None
   Active chloroplasts	None	None	1.0 ml	1.0 ml	1.0 ml
   DCMU	None	None	None	None	1.0 ml
   Water	2.0 ml	1.0 ml	1.0 ml	1.0 ml	None

4. Turn on the spectrophotometer and adjust the wavelength to 600 nm. Use the solution in tube #1 to blank the spectrophotometer.

5. Add 1.0 ml of DCPIP dye to tube #2, mix by swirling and immediately measure the absorbance at 600 nm.

6. Add 1.0 ml of DCPIP dye to tubes #3, #4 and #5. Mix and immediately measure the absorbance of each. Note the time for each reading.

7. Wrap tube #4 in dark paper, and aluminum foil. This tube serves as a "dark" control and the absorbance of this solution will be read only at the end of the experiment. It is important that no light reach the chloroplasts inside.

8. Place tubes #2, #3, and #5 in a beaker of ice water and illuminate with a strong light source. 5

9. Measure the absorbance of each tube after 10 minutes, 20 minutes and finally after 30 minutes. Be sure to wipe the tubes dry before inserting the tube into the spectrophotometer. Record your results.

   Time (Minutes)	2	3	4	5
   0
   10
   20
   30

10. Calculate the total dye reduced at 10 minute intervals using the equation:

    		Co(Ao-Ac)
    Cr	=	-----------
    		Ao

    where:
    Cr is the dye reduced in moles/liter
    Co is the original dye concentration in moles/liter
    Ao is the absorbance of the mixture at the start
    Ac is the absorbance of the mixture at the end of the experiment.

11. Calculate the percent dye reduced and plot percent dye reduced per minute for tubes 2 through 5.

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    Cell Biology Laboratory Manual

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    Dr. William H. Heidcamp, Biology Department, Gustavus Adolphus College,
    St. Peter, MN 56082 -- cellab@gac.edu